Reactive oxygen species (ROS)--inevitable intermediates arising from aerobic metabolism, including hydrogen peroxide, superoxide ion, and hydroxyl radical--affect a number of cellular events, ranging from cell growth and aging to cell death. Understanding how ROS affect cellular processes will greatly enhance our basic knowledge of biology and facilitate medical research in various fields. My research during the past year focused on the protein targets through which ROS exert their influence. (1) We found two proteins in A431 cells that receive heavy glutathionylation upon hydrogen peroxide treatment. Contradictory to the observation that growth factor treatment increases cellular hydrogen peroxide levels, treatment of cells with growth factor led to de-glutathionylation of the proteins. Therefore, these proteins may play important roles in the regulation of cell growth. Current efforts are focused on the purification and characterization of these proteins. (2) ROS have different effects on different protein targets. It has been reported that superoxide radical ion deactivates PTP1B (an important signal transduction enzyme) much faster than hydrogen peroxide does. To study the physiological relevance of ROS on protein targets, we compared the inhibition effects of different ROS on PTP1B and other active-site thio-containing proteins to quantitate those effects. (3) Perhaps the most direct way to study the effects of ROS on cell pathways is to introduce ROS into cells and then study cellular changes. Similarly, opposite effects can be observed when ROS scavengers (catalase, SOD, etc.) are introduced into cells to remove cellular ROS. We constructed DNA plasmids encoding human catalase and Cu,Zn-SOD and transfected A431 and NIH3T3 cells with these plasmids. The protein expressions, however, are not as stable as expected. On the other hand, we found that these proteins can be introduced into cells in large quantities by electroporation. Using this method, we are studying how ROS affect cell growth and programmed cell death. - ROS, catalase, SOD, hydrogen peroxide, superoxide, glutathionylation